The identification of unique DNA or RNA sequences or specific genes within a biological sample may indicate the presence of a physiological or pathological condition, such as cancer or a pathogen infection. For example, nucleic acid hybridization assays may be used in the agriculture and food processing industries to detect plant pathogens or harmful bacteria.
Luminescent labels, which emit light upon excitation by an external energy source, have proven useful to detect nucleic acid molecules and to probe the interaction between these molecules. These labels are categorized by the source of the exciting energy, such as, for example: (1) photoluminescent or fluorescent labels, which are excitable by units of electromagnetic radiation of infrared, visible or ultraviolet light; (2) chemiluminescent labels, which obtain energy from chemical reactions; (3) radioluminescent labels, which are excitable by energy from high energy particles; and (4) bioluminescent labels, which are excitable by energy supplied in a biological system. The use of luminescent labels permits “homogeneous” assay techniques in which a probe labeled with a luminescent label exhibits different luminescent characteristics when associated with a target, thereby obviating the need to remove unassociated labeled probe. See, e.g., Morrison et al., Anal. Biochem. 183: 231 (1989).
Luminescent labels also have proven useful in nucleic acid amplification techniques. Luminescent signal primers (also referred to as “detector probes”), which hybridize to a target sequence downstream of the hybridization site of amplification primers, have been described for use in detection of nucleic acid amplification. See, e.g., U.S. Pat. No. 5,547,861, which is incorporated by reference herein. A signal primer is extended by polymerase in a manner similar to extension of the amplification primers. Extension of the amplification primer displaces the extension product of the signal primer in a target amplification-dependent manner, producing a detectable double-stranded secondary amplification product that indicates target amplification. The secondary amplification products generated from signal primers may be detected by various means, as exemplified in U.S. Pat. No. 5,550,025 (incorporation of lipophilic dyes and restriction sites) and U.S. Pat. No. 5,593,867 (fluorescence polarization detection), which are incorporated by reference herein.
Fluorescence energy transfer has been used advantageously to probe the interaction between complementary nucleic acids in various hybridization methods. Fluorescent energy transfer occurs between a donor fluorophore and a quencher molecule (which may or may not be a fluorophore) when the absorption spectrum of the quencher overlaps the emission spectrum of the donor and the two are in sufficiently close proximity. The excited-state energy of the donor is transferred by a resonance dipole-induced dipole interaction to the neighboring quencher, resulting in quenching of donor fluorescence. In some cases, if the quencher is also a fluorophore, the intensity of its fluorescence may be enhanced. The efficiency of energy transfer depends on the distance between the donor and quencher in a manner well known in the art.
Probes or primers comprising intramolecularly base-paired secondary structures capable of exhibiting changes in fluorescence quenching recently have been employed for detection of nucleic acid target sequences. In these systems, a donor and a quencher fluorophore, which are in close proximity in the base-paired secondary structure of the probe or primer, become spatially separated due to unfolding of the secondary structure because of base-pairing with a target. The target-dependent separation of the two fluorophores reduces quenching of the donor fluorophore, which increases the fluorescence intensity of the donor. For example, a self-complementary oligonucleotide labeled with fluorophores at its 5′ and 3′ ends forms a hairpin that brings the two fluorophores into close proximity so that energy transfer and quenching can occur. Hybridization of the self-complementary oligonucleotide to its complement on a target oligonucleotide disrupts the hairpin and increases the distance between the two fluorophores, thereby reducing quenching. Hairpin structures labeled in this manner are described by Tyagi et al., Nature Biotech. 14: 303-308 (1996), for example.
A disadvantage of the hairpin structure is that it is very stable, and conversion to the unquenched, hybridized form is often slow and only moderately energetically favored. In the case of the structures disclosed in Tyagi et al., supra, for example, the target sequence, must compete for hybridization with the complementary sequence that forms the hairpin structure, thereby lowering performance.
A partially single-stranded, partially double-stranded signal primer labeled with a donor/quencher pair has also been described. For example, EP 0 878 554 discloses a signal primer with a donor/quencher pair flanking a single-stranded restriction endonuclease recognition site. In the presence of the target, the restriction site becomes double-stranded and cleavable by a restriction endonuclease. Cleavage separates the donor/quencher pair to decrease donor quenching.
Other methods have employed restriction endonuclease sites to separate a donor/acceptor pair. Japanese Patent No. 93015439 B discloses methods for measuring polynucleotides by hybridizing a single-stranded target to a single-stranded polynucleotide probe labeled with an energy transfer pair. The double-stranded hybrid is then cleaved between the labels by a restriction enzyme, and fluorescence of one of the labels is measured. A disadvantage of this method is that the restriction site in the probe must also be present in the target sequence being detected. Ghosh et al., Nucl. Acids Res. 22: 3155-3159 (1994) describe restriction enzyme-catalyzed cleavage of a fluorophore-labeled oligonucleotide, which is analyzed using fluorescence resonance energy transfer. In this assay, a labeled oligonucleotide is hybridized to a complementary sequence that also is labeled to produce a cleavable double-stranded restriction site. A disadvantage of this method is the requirement to modify each of the two strands with a fluorescent label.
A donor/acceptor pair also has been used in amplification methods, such as a strand-displacement amplification (SDA) method described in U.S. Pat. Nos. 5,919,630; 5,846,726; and 6,054,729, which are herein incorporated by reference in their entirety. These patents disclose a single-stranded signal primer that is modified by linkage to a donor/acceptor pair. The signal primer may further comprise a restriction endonuclease recognition site between the donor and acceptor. When the signal primer forms a duplex with a complementary target sequence, the restriction endonuclease recognition site is rendered double-stranded and cleavable or nickable by a restriction endonuclease. Cleavage or nicking separates the donor and acceptor, and a change in fluorescence due to decreased quenching is detected as an indication of target sequence amplification.
There remains a need for improved methods for detection of nucleotide sequences. Specifically, there is a need to improve the ratio between a fluorescence signal indicative of target amplification and a background signal created by light scattering and non-quenched fluorescence. There also is a need for more efficient means of amplification detection and greater sensitivity of fluorescent detection methods.